The overall objective of this project is to elucidate the mechanisms that control the activation of caspase-9. Caspase-9 is a major initiator caspase in mammalian cells. It exists in healthy cells as a latent precursor and becomes activated in response to a wide range of cell-intrinsic apoptosis stimuli such as developmental lineage information, oncogene activation, DNA damage, and nutritional deprivation. The intrinsic apoptosis pathway that activates caspase-9 is the major mechanism by which the formation of tumor cells is prevented in the first place, and by which tumor cells are killed by chemo- and radiation therapy. The activation of caspase- 9 occurs in a large cytosolic complex known as the apoptosome, the formation of which is triggered by the release of mitochondrial cytochrome c to the cytosol and the subsequent binding of cytochrome c to Apaf-1. This binding enables Apaf-1 to assemble into the heptameric apoptosome, which recruits procaspase-9 and permits its auto-activation. Previous studies by us and others demonstrated that the activation of initiator caspases is induced by their oligomerization. We will determine the mechanism for oligomerization-induced procaspase-9 activation (Aim 1). In addition, we found that tRNAs inhibit caspase-9 activation through direct interaction with cytochrome c and preventing the formation of the apoptosome. We will determine this important function of tRNAs which is beyond their well-established role in the transmission of genetic information. We will also examine this function of tRNAs in apoptosis resistance of tumor cells (Aim 2). Finally, the activation of caspase-9 in the apoptosome is inhibited by the inhibitor of apoptosis proteins (IAPs). This inhibitory effect of IAPs is countered during apoptosis by mitochondrial IAP-binding proteins. Compounds mimicking the effect of IAP-binding proteins show promise in tumor therapy. We recently identified a novel IAP- interacting protein residing in mitochondria. We will elucidate the role of this protein in caspase-9 activation (Aim 3). These three aims represent a concerted effort to elucidate the intricate control of caspase-9 activation at multiple levels. We believe that they will help the development of more effective treatments for apoptosis- related diseases, particularly cancer.